Photoreceptor guanylate cyclases (GC1 and GC2) produce cGMP, the internal messenger of phototransduction. Cations, mediated by Ca2+-binding proteins termed guanylate cyclase activating proteins (GCAPs), regulate their activities. Mutations affecting Ca2+-binding in the GCAP1 gene (GUCA1A) have been linked to autosomal dominant cone dystrophy (adCD) and autosomal dominant cone-rod dystrophy (adCORD). The biochemical defects present as a dominant, persistent stimulation of GC1 at dark [Ca2+]. In specific aim 1, we will characterize animal models of adCD and adCORD based on GCAP1 mutations, and develop an RNA interference strategy to knock down both the mutant and the normal GCAP1 genes. A partial or complete knockdown will delay onset or cure the cone dystrophy since GCAP null mice have only a minor phenotype (delay in dark adaptation) and no retinal degeneration. These experiments are a prelude to eventual human gene therapy. In specific aim 2, we propose to generate a GCAP1 knockout mouse by transgenic expression of GCAP2 on a GCAPs knockout background to investigate the kinetics of recovery in the absence of GCAP1. Further, we will explore the biochemical consequence of a GCAP2(G157R) mutation linked to dominant RP, first in-vitro and finally in a transgenic experiment. In specific aim 3, we propose to generate mice expressing GC1 only or GC2 only on a GCAPs knockout background to dissect the contribution of each enzyme to the dark current in the absence of Ca2+ stimulation. We further propose to generate transgenic mice expressing GC2 in mouse cones using a cone specific promoter. The purpose is to characterize the physiology of cones expressing GC2 but not GC1,and the Ca2+ sensitivity of GC2 in the presence of GCAP1 (which poorly stimulates GC2 in vitro). While analyzing the phenotypes of GC1 and GC2 knockouts in mouse, we discovered that both GCs, as integral membrane proteins, have key roles in transport of membrane-associated proteins (PDE, GRK1, Transducin) to the outer segments. We therefore propose to identify the targeting sequences in transgenic Xenopus using constructs expressing EGFP, a rhodopsin C-terminal segment lacking the targeting sequence VXPX, and C-terminal GC fragments containing the proposed targeting motif(s). The proposed research will contribute to our understanding of the cGMP/Ca2+ feed back system in photoreceptors, as well mechanisms leading to retina disease based on GCAP mutations.